The Modular Architecture Of The Saccharomyces Cerevisiae Mediator Complex

Mediator,a large complex highly conserved throughout eukaryotes,is preeminently responsible for transcription pre-initiation complex(PIC)assembly in vivo and for conveying regulatory signals from DNA-binding transcription factors to PIC.Although Mediator plays an essential and critical role in the regulation of transcription in all eukaryotes,the detailed architecture and molecular mechanisms of the complex remain largely elusive.Mediator subunits have been suggested to be organized into four functional modules,Head,Middle,Tail and dissociable CDK8 kinase modules,the modular architecture and subunit composition are conserved from yeast to human.Cryo-EM is a powerful technique for structural characterization of macromolecular complexes in their close to native state,which can characterize macromolecular complex in distinct functional states and determine structural models for transient functional states.Although the EM structures of the Mediator complex from several species have been determined,none of the subunits has yet been clearly localized in the complex by EM.Due to the complexity,flexibility and modularity of the core Mediator,many basic questions,such as the boundaries of the three modules,how they are organized,and how they interact with each other,remain unresolved.To understand how the Mediator carries out multiple essential functions,it is critical to characterize the modular architecture,and to understand how the modules are organized and how they interact with each other to enable regulation of transcription.Firstly,we improved the biochemical preparation of the core Mediator complex and the homogeneity and concentration were substantially improved.Three different conformations were reconstructed and the base domain pivoted on a central connection to the upper portion.To increase the resolution of the Mediator structure,we divided Mediator into modules or module assemblage and characterize the structures and functions individually.We compared the EM structures of the core Mediator and its subcomplex,especially the first3D structure of the Head+Middle modules,performed nanogold labeling of multiple subunits from different modules,and proposed a new modular organization of Mediator,which resolves many inconsistencies of the previous model with the biochemical and functional studies.The redefined model allowed us to integrate the structural and functional information into a coherent mechanism for Mediator modularity and regulation in transcription initiation.We studied in details the substantially conformational and compositional heterogeneity of the Head+Middle modules,and illuminate the interaction surface between Head and Middle modules.Moreover,we reconstructed the Head module structure in different conformations.The?20%amino acid residues of Head module missing in the crystal structure could be resolved in the EM structures.Furthermore,we found Head module is the scaffold for the Mediator and Middle is the most flexible and dynamic module.Although increasing evidence demonstrates that CDK8 module plays both positive and negative roles in transcription regulation,the detailed structure,and subunit organization,molecular mechanism how it regulates transcription remain elusive.We used single-particle electron microscopy to characterize the structure and subunit organization of the CDK8 module and illuminated the substantial mobility of the Medl3 subunit results in the structural flexibility.The CDK8 module interaction with core Mediator is concurrent with active transcription in vivo.An interaction with the CDK8 module induces core Mediator into much extended conformation in vitro,which is presumed to be an active functional state of Mediator.These results illuminated the detailed architecture of Cdk8 module,and suggested the CDK8 module could positively regulate transcription by modulating Mediator conformation.Taken together,our cryo-EM studies on Mediator laid solid foundation to illuminate its module architecture and to understand how the Mediator carries out multiple essential functions in eukaryotic transcription.